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First published online 3 March 2009
doi: 10.1242/jcs.028183

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Cell polarity triggered by cell-cell adhesion via E-cadherin

¹ Department of Bioengineering, University of Pennsylvania, Philadelphia, PA 19104, USA
² Department of Biomedical Engineering, Johns Hopkins University School of Medicine, Baltimore, MD 21205, USA

View larger version (43K): [in this window] [in a new window]   Fig. 1. Cell-cell contact polarizes the cell-motility apparatus. (A) Cells seeded in 10% serum at subconfluence or confluence were switched to 0% or 10% serum-containing media and imaged 33 hours later (5 hours after the scrape wound). (B) Cells 4 hours after scrape wounding. (C) Edge cells were scored as being oriented if their centrosome was distal from cell-cell contacts relative to the nucleus. (D) Kinetics of orientation. Broken line indicates random (33%) orientation; t=0 corresponds to time of scrape wounding. (E) Cells in a patterned monolayer 24 hours after plating. Inset is a 250,000 µm2 patterned monolayer and shows the region of the main panel. (F) Edge orientation as in C. (G) Kinetics of orientation as in D, except that t=0 corresponds to cell plating. (H) Cells on patterns 24 hours after plating. Boxed areas are shown at higher magnification on the right of each panel. (I) Cells in a paired configuration 24 hours after plating. (J) Orientation as in C. (K) Kinetics of orientation as in D, except that t=0 corresponds to cell plating and the broken line indicates random (50%) orientation. (L) Cells in a paired configuration 8 hours after plating. (M) Pattern (gray), nuclear (blue) and centrosomal (green) positions were measured 12 hours after plating from micrographs of 50 patterns from three experiments. (N) Frequency histogram of organelle locations. In D, G and K, means ± s.e.m. are from at least three independent experiments. Scale bars: 25 µm.

View larger version (41K): [in this window] [in a new window]   Fig. 2. Cell-cell contact polarizes cell dynamics. (A) Representative image and kymographs of cell membranes that are distal (i) or proximal (ii) to the cell-cell contact from a 15-minute time-series. In the kymographs, vertical and horizontal bars are 10 µm and 60 seconds, respectively. (B, left) Activity map of the cell pair in A. (B, right) Dynamic activity per pixel of membrane that is proximal (dashed boxes in the left image) or distal (solid box in the left image) to the cell-cell contact for 20 cells. Means (horizontal bars) ± s.e.m. (gray boxes) are indicated (*P=0.0112; Student's t-test). (C) Representative time series of a pair of cells after release (time indicated) from patterned constraints (broken lines) 20 hours after plating. Scale bars: 25 µm unless noted.

View larger version (49K): [in this window] [in a new window]   Fig. 3. E-cadherin is required for cell-cell-contact-induced polarity. (A) Cells were switched to minimum essential medium modified for suspension cultures (SMEM) containing 1.8 or 0 mM Ca2+ at 4 hours, and fixed at 24 hours, after plating; processed as in Fig. 1I-K (*P=0.018; Student's t-test). (B,C) Cells infected with Ad-GFP or Ad-E at least 6 hours prior to, and fixed 24 after, plating; processed as in Fig. 1I-N (**P=0.0032; Student's t-test). (D) Representative images and kymographs from a 15-minute time series. In the kymographs, vertical and horizontal bars are 10 µm and 60 seconds, respectively. (E) Activity maps of the cell pairs in D, and dynamic activity per pixel of membrane that is proximal (dotted boxes) or distal (solid box) to the cell-cell contact for 20 cells (Ad-GFP, green) and nine cells (Ad-E, blue). Means ± s.e.m. are indicated (*P<0.05; ns, P>0.05; ANOVA and Tukey's HSD). (F,G) Cells were infected at least 24 hours prior to wounding and imaged subsequently (F) or immunolabeled and scored 4 hours after wounding (G) (ns, P=0.45; **P=0.0031; Student's t-test). The broken line indicates random orientation, and means ± s.e.m. are from at least three independent experiments. Scale bars: 25 µm unless noted.

View larger version (46K): [in this window] [in a new window]   Fig. 4. Polarity triggered by cell-cell contact requires the actin cytoskeleton and Cdc42. (A-D) G0-synchronized cells were treated with 1% DMSO or 1 µM cytochalasin D (cyto. D) at 3 hours, and fixed at 8 hours, after plating; processed as in Fig. 1I-N (**P=0.0072; Student's t-test). (E) Cells were infected with Ad-GFP or Ad-RhoN19 at least 12 hours prior to, and fixed 24 hours after, plating; processed as in Fig. 1I-K (ns, P=1; Student's t-test). (F) Cells were handled as in A, except that they were treated with PBS or 50 µM NSC23766 (ns, P=0.17; Student's t-test). (G) Representative immunoblot and quantification of total and GTP-bound Cdc42, measured via pull-down assay from cells transfected with 10 nM siRNA (*P<0.05; ns, P>0.05; ANOVA and Tukey's HSD). (H-J) siRNA-transfected cells fixed 8 hours after plating and processed as in Fig. 1I-N (*P<0.05; ns, P>0.05; ANOVA and Tukey's HSD). Broken lines in C, E, F and I indicate random orientation, and means ± s.e.m. are from at least three independent experiments. Scale bars: 25 µm.

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